1. Field of the Invention
The invention in the field of microbiology, immunology and medicine relates to novel proteins, peptides and antigenic compositions for early diagnosis of mycobacterial disease, kits comprising such compositions, and serodiagnostic methods using such compositions.
2. Description of the Background Art
Recent World Health Organization data show that the global incidence of tuberculosis (TB) is increasing at 1% per year and that there are an estimated 8.8 million new cases each year (1). One concerning fact is less than half of the 8.8 million estimated cases are diagnosed as smear positive. This underscores the need for a rapid, sensitive diagnostic test to aid TB control efforts. The development of such a test has proven to be one of the greatest challenges in TB research.
In recent years there has been renewed interest in developing antibody-based diagnostics that utilize multiple antigens to achieve high levels of sensitivity and specificity (2). The success of a serodiagnostic test for TB hinges on its ability to detect multiple disease categories, including pauci- and multibacillary forms, pediatric cases, and patients co-infected with human immunodeficiency virus (HIV). Previous work from our laboratories identified several antigens that provide high sensitivity and specificity when used in an ELISA format (3-9). Furthermore, this work highlighted differential antigen reactivity based on the disease category (3, 5, 6, 8-10). However, a complete analysis of patients' serological reactivity to a large proportion of the Mycobacterium tuberculosis (Mtb) proteome is hindered by the inability to evaluate the reactivity of the nearly 4,000 predicted proteins of Mtb in a high-throughput fashion.
Over the past several years, protein microarrays have shown considerable potential for detecting antigen-antibody interactions on a proteomic scale (11-13). As a proof-of-principle, Bacarese-Hamilton, et al. (14) immobilized recombinant antigens of various pathogens to glass slides, and human antibodies specific for each antigen were detected in sub-picogram amounts. This microarray assay also performed at the same level of efficiency as conventional ELISA-based methods in differentiating between positive and negative sera. Microarray technology has now been extended to characterize antibody responses generated upon vaccination with Yersinia pestis (15) and vaccinia virus (16). Protein microarrays accommodate thousands of individual antigens or antigen pools on a single slide, and automation allows for hundreds of slides to be generated at once. Moreover, this methodology is facile and allows reproducible screening of sera from a large number of individual patients.
A critical element of tuberculosis control is early and sensitive diagnosis of infection and disease. The present inventors' laboratories recently showed that different stages of disease were distinguishable via two-dimensional western blot analyses of Mycobacterium tuberculosis culture filtrate proteins (CFPs). However, this methodology is not suitable for high-throughput testing. Advances in protein microarray technology provide a realistic mechanism to screen a large number of serum samples against thousands of proteins to identify biomarkers of disease categories. Using such an approach, the present inventors identified four Mtb proteins, and peptides thereof as being of particular value for serodiagnosis of tuberculosis at both early and later stages of the disease.